Abstract
Cobalamin-independent methionine synthase (MetE) catalyzes the transfer of a methyl group from methyltetrahydrofolate to L-homocysteine (Hcy) without using an intermediate methyl carrier. Although MetE displays no detectable sequence homology with cobalamin-dependent methionine synthase (MetH), both enzymes require zinc for activation and binding of Hcy. Crystallographic analyses of MetE from T. maritima reveal an unusual dual-barrel structure in which the active site lies between the tops of the two (βα)8 barrels. The fold of the N-terminal barrel confirms that it has evolved from the C-terminal polypeptide by gene duplication; comparisons of the barrels provide an intriguing example of homologous domain evolution in which binding sites are obliterated. The C-terminal barrel incorporates the zinc ion that binds and activates Hcy. The zinc-binding site in MetE is distinguished from the (Cys)3Zn site in the related enzymes, MetH and betaine–homocysteine methyltransferase, by its position in the barrel and by the metal ligands, which are histidine, cysteine, glutamate, and cysteine in the resting form of MetE. Hcy associates at the face of the metal opposite glutamate, which moves away from the zinc in the binary E·Hcy complex. The folate substrate is not intimately associated with the N-terminal barrel; instead, elements from both barrels contribute binding determinants in a binary complex in which the folate substrate is incorrectly oriented for methyl transfer. Atypical locations of the Hcy and folate sites in the C-terminal barrel presumably permit direct interaction of the substrates in a ternary complex. Structures of the binary substrate complexes imply that rearrangement of folate, perhaps accompanied by domain rearrangement, must occur before formation of a ternary complex that is competent for methyl transfer.
Highlights
Methionine synthases catalyze the transfer of a methyl group from N5-methyl-5,6,7,8-tetrahydrofolate (CH3-H4folate) to L-homocysteine (Hcy), the terminal step in the biosynthesis of methionine
In8 barrel enzymes, the active site is usually located near the C-termini of the inner barrel strands, with catalytic residues contributed by the b–a segments that join these strands to the outer helices
In the complexes of A. thaliana methionine synthase (MetE) with Hcy or methionine, the distances between zinc and substrate or product sulfur are long, as is the case in the Hcy complex of MetE from T. maritima, but water rather than glutamate has been assigned as the ligand opposite to Hcy or Met
Summary
Methionine synthases catalyze the transfer of a methyl group from N5-methyl-5,6,7,8-tetrahydrofolate (CH3-H4folate) to L-homocysteine (Hcy), the terminal step in the biosynthesis of methionine. MetH and MetE both face the same mechanistic challenge They must catalyze the transfer of a very poor leaving group from the tertiary amine, CH3-H4folate, to a relatively poor nucleophile, the sulfur of Hcy. MetH facilitates this transfer by using cobalamin as an intermediate methyl carrier [1]. MetE appears to catalyze the direct transfer of the methyl group from CH3-H4folate to Hcy [3]. This latter strategy seems to offer a less satisfactory answer to the mechanistic problems: measured kcat values for MetE are smaller than those for MetH by a factor of approximately 50–100
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